The invention relates to the identification of infectious pathogens, particularly viruses, bacteria and other microorganisms.
The invention provides a method whereby pathogens of acute infections can be identified, without first culturing them in external nutrient media, by mass spectrometric measurement of their protein profiles obtained from pathogens directly precipitated from body fluid into pellets by centrifuging. With this method, pathogens which cause acute infections can be identified in less than one hour.
Many kinds of microorganism (which will also be referred to below as microbes), particularly bacteria and unicellular fungi, are very easy to identify by a recently introduced mass spectrometric process in which small quantities of microbes from a colony cultivated in the usual way in a nutrient medium are transferred to a mass spectrometric sample support plate, and then subjected directly to mass spectrometric analysis. The mass spectrum especially shows the different proteins, provided they are present in the microbes in sufficient concentration. The identity is then determined from the microbe's protein profile through reference to spectral libraries containing thousands of reference spectra.
The nutrient medium is usually contained in moist gelatine in a Petri dish, and separate colonies of pure strains are obtained from swabs in the usual way by culturing in about six to twenty hours, depending on the growth rate of the microbes. If the colonies overlap or become mixed, it is possible to obtain pure colonies, also in the usual way, by culturing a second time. A quantity of microbe culture is transferred with a small spatula, stick or pestle from a selected colony onto the mass spectrometric sample support plate and a solution of a conventional matrix substance for ionization by matrix assisted laser desorption (MALDI) is sprinkled on. The organic solvent in the matrix solution usually penetrates into the microbe cells and destroys them by osmotic force. The sample is then dried by evaporating the solvent, leading to crystallization of the dissolved matrix material. Soluble proteins and also, to a small extent, other cell substances become incorporated as analyte molecules into the matrix crystal.
The matrix crystals with the embedded analyte molecules are exposed to pulsed laser light in a mass spectrometer, which generates ions of the analyte molecules. The analyte ions can then be measured according to their mass in the mass spectrometer. Time-of-flight mass spectrometers are used preferably for this purpose. The mass spectrum is the profile of mass values and intensities of these analyte ions. Protein ions predominate; the most useful information is found in the mass range between about 3,000 and 15,000 daltons. In this method, practically all protein ions carry a single charge only (number of charges z=1), which means that it is possible here simply to speak of the mass m of the ions rather than always using the term “mass-to-charge ratio”, m/z, as is otherwise usual—and necessary—in mass spectrometry.
This protein profile is highly characteristic of the particular microbe because every species of microbe produces its own, genetically programmed proteins, each with a characteristic mass. The protein profiles are characteristic for microbes in rather the same way as fingerprints are for people. Reliable libraries of mass spectra of the protein profiles of microbes, suitable for medical and legal applications (so called “validated libraries”), are nowadays being developed with cooperation from many sites, including diagnostics companies, university institutes, hospitals, and national institutes.
This identification method has proved to be extraordinarily successful. The certainty of correct identification is much greater than was possible with the microbiological identification methods used in the past. It has been demonstrated that the reliability of identification is well over 95 percent for hundreds of different kinds of microbes. However, it proved difficult to determine the reliability properly because the microbes from the known collections have been wrongly identified in more than only a few cases. In the end, only genetic sequencing can help to put the identification beyond any doubt and this has confirmed the mass spectrometric identification in the great majority of cases.
In many cases, this simple procedure even makes it possible to distinguish closely related strains of the same species of microbes, as the proteins present in microbes are genetically programmed and can vary distinctly between strains. Small changes in the genetic blueprint necessarily result in proteins with a different structure and masses that differ from genetically unmodified proteins; they therefore yield a different protein profile, provided the concentration of the proteins with changed mass in the microbes is sufficient to produce a signal strong enough for mass spectrometric analysis. It has already been possible to correct taxonomic classifications and relationships of microbes in this way.
If no reference mass spectrum is present in a library for the precise species of microbe being examined (which often happens, due to the hundreds of thousands of microbe species and the limited size of spectrum libraries so far available), library searches with looser similarity requirements can provide at least some indication of the order, family or genus of the microbes, since related microbes frequently contain a number of identical protein types.
For the protein ions of identical microbe species, the masses are, by their nature, always identical and therefore strictly reproducible, but the intensities of the protein signals reproduce only approximately. The use of different nutrient media for the culture has an effect on the metabolism of the microbes and therefore on the creation of the different proteins in varying proportions and so on their concentration and their intensity in the protein profile. The effect is, however, not strong. The variations in intensity do not interfere with the identification, provided a suitable computer program is used. Equally, the maturity of the colonies has an effect on the relative intensities of the protein signals in the mass spectra and here again only to a small extent. Characteristically different mass spectra from the same species of microbe are in fact only found in the case of microbes which can adopt different life-forms, such as spore-formers: the spores exhibit different protein profiles from the normal cells. If freshly cultured microbes are used, however, this difference is not important.
Computer programs for searching libraries and performing spectral comparisons can cope with variations in intensity, which only play a subsidiary role here. As has been noted above, the identification of microbes with these programs is very reliable. The programs operate without identifying the individual proteins involved (which could be done by means of fragment ion spectra), only using the similarity of the mass spectra. In the similarity search, masses are regarded as highly significant and intensities as much lower significant. It is even possible for some proteins to be absent from the mass spectra (due to very low intensity) without interfering with the similarity determination: matching the mass values for a large majority of proteins is enough for identification. Usually the library spectra store information as to which protein signals must always be present, for instance, by storing thresholds for the intensities, or information about the probability of finding the protein signals under observation in frequently repeated spectral recordings of different samples.
The reference spectra in the spectral libraries can, for instance, contain the masses, mass tolerances, mean intensities, standard deviations of the intensities, and appearance probabilities of the individual protein signals. The reference spectra are generally obtained from frequently performed raw measurements, preferably from different cultures, by automatic computer evaluation; they can also, however, be reduced by including additional knowledge about the microbes (see, for instance, the patent publications DE 100 38 694 A1 and DE 103 00 743 A1, W. Kallow et al.).
The method briefly described above, in which a few microbes from a colony are smeared onto a reserved spot on a mass spectrometric sample support, followed by sprinkling with matrix solution, is the simplest method of sample preparation and, so far, the fastest. The process can also be automated with the aid of image-recognizing pi-petting robots for use in routine laboratories. After culturing a colony that is only just visible, it only takes one or two hours to achieve full identification, even if hundreds of samples have to be analyzed at the same time. Mass spectrometric sample support plates for 96 or 384 samples are commercially available; it takes between about half an hour and two hours to record these mass spectra. For rush jobs, individual microbe samples can be identified within a few minutes, although culturing, which always takes time, is necessary first.
Other methods of sample preparation have also been investigated, such as extraction of the proteins after destroying the microbes ultrasonically in a tube, or extraction methods for the proteins after dissolving the sometimes resistive cell walls using strong acids. These methods of decomposition are used when the normal method of smearing the microbes fails due to the microbe cell walls not being destroyed when the matrix solution is sprinkled on. If the normal method yields enough good mass spectra for a comparison, the decomposition methods give results that are very similar to the simple smearing method, but do often exhibit clearer mass spectra with less background interference. Both methods yield mass spectra which can identify the microbes using the same library of reference mass spectra.
Today, the mass spectra of the microbe proteins are recorded using time-of-flight mass spectrometers operating in linear mode, due to the particularly high detection sensitivity, although the mass resolution and mass accuracy of the spectra from time-of-flight mass spectrometers operating in reflector mode is significantly better. In reflector mode, however, only around a twentieth of the ion signals appear and the detection sensitivity is up to two orders of magnitude lower. The reason for the high sensitivity is that, when a time-of-flight mass spectrometer is operating in linear mode, not only the stable ions are detected, but also the fragment ions generated by “metastable” ion decay. Secondary electron multipliers are used to measure the ions, as a result of which even neutral particles created en route as a result of ion decay are measured by the ion detector, since they also generate secondary electrons on impact. All of these fragment ions and neutral particles that have been created from one parent ion species have the same velocity as the parent ion and therefore reach the ion detector at the same time. The time of arrival is a measure of the mass of the original intact ions.
For many applications, the higher detection sensitivity is of such importance that many of the disadvantages of linear operation of the time-of-flight mass spectrometer, such as a significantly lower mass resolution, are accepted. For these applications, the energy of the desorbing and ionizing laser is increased, raising the ion yield but also decreasing their stability, although that is not of great importance here.
Recording mass spectra with time-of-flight mass spectrometers generally requires a large number of single spectra to be recorded and digitized in rapid succession; usually producing a sum spectrum by adding together measurements with the same flight time. The ions for each individual spectrum are generated by a shot from a pulsed UV laser. This method of generating sum spectra is necessary because of the low dynamic range in an individual spectrum. A minimum of 50 and in some cases even 1,000 or more individual spectra are recorded; a sum spectrum generally consists of several hundred individual spectra, and these can be recorded and added together within a few seconds in modern mass spectrometers. The total time required to record a sum spectrum depends on the number of individual spectra and on the firing frequency of the laser being used. Lasers firing at between 20 and 200 hertz are in use nowadays for this purpose, but other electronics as, for instance, delayed acceleration voltages also must be switched correspondingly, determining the applicability of faster lasers. If medium-speed processes are used, somewhere between 2 and 30 seconds are required to record a good sum spectrum.
In the fields of application mentioned above, mass spectra extending from about 1,000 daltons up to the high mass ranges of, for instance, 20,000 daltons are measured. It has been found that the mass signals in the lower range of masses up to about 2,500 daltons cannot be effectively evaluated, as they originate from externally attached non-specific peptides and other substances whose presence tends to be random and variable. For microbes smeared onto the sample support plate, the best identification results are obtained when only the mass signals in the range between about 3,000 and 15,000 daltons are evaluated. For microbes decomposed after thorough cleaning in centrifugation tubes and transfer of the decomposition liquid to a sample plate with pre-prepared thin layer matrix, the mass range from 1000 to 15000 daltons may used since the resulting spectra are much clearer and better reproducible in the lower mass range.
For the reasons of low mass resolution mentioned above, isotope groups cannot be resolved in these mass ranges. The isotope groups consist of ion signals that differ by only one dalton. Only the envelopes of the isotope groups are measured. However, mass spectrometric measurement methods that offer a higher resolution and higher mass accuracy are also known; but it is not yet known whether comparable sensitivities can be achieved with them.
This method for identifying microbes generally requires a pure culture of microbes in order to obtain a mass spectrum that is not overlaid by the signals of other microbes. It has, however, been found that mass spectra from mixtures of two microbe species can be evaluated, and that both species of microbe can be identified. The reliability of the identification is only slightly affected. If more than two microbe species are included in the mass spectrum, the probability and reliability of identification decrease very sharply.
The method for simply identifying microbes by mass spectrometry can find applications in many fields, such as the monitoring of drinking water or quality control in food manufacture. In food manufacture, the species of microorganisms present are crucial to whether the food can be consumed without risk. We need only think of harmful staphylococci, streptococci or salmonella, which must be detected through ongoing checks. On the other hand, beer, wine, cheese and yoghurt could not be created without the useful work of billions of microbes. Purity of the strains is crucial for their use.
Particularly strict and reliable monitoring is required in the medical field. Pathogens must be kept away from hospitals. Constant monitoring of the microbes, and their identification, is a strict statutory requirement for operating rooms, for example.
The identification of microbes involved in infectious illnesses plays a particular role. Here it is important to be able to identify the pathogens very quickly so that the correct medical intervention can be taken immediately. In spite of the need to first grow microbe cultures, the method of mass spectrometric identification is one or two days faster than the microbiological methods used up to now. Nevertheless, it still takes about 12 to 24 hours, and the time can be significantly longer if a second culturing happens to be necessary. For many applications, particularly in the case of acute infections, this time is too long, and the search for faster procedures is urgent.
In WO 2002/021,108 A2 (N. G. Anderson and N. L. Anderson), a method is presented to extract, separate, and purify microbes including viruses by twodimensional ultra-centrifuging directly from body fluids or homogenized tissue. In a first centrifuging step, all particles are removed having a sedimentation speed higher than those of the microbes to be identified. In the second ultra-centrifuging step, isopycnic banding is used in liquids filled in to form a wide-range density gradient, using special serrated centrifuge tubes. The microbanded microbes can be recognized and taken out by complex apparatuses, washed, even centrifuged after washing once more to form pellets, in order to prepare samples containing one kind of microbes only for different types of analysis procedures. “Once the viruses from a biological sample have been highly purified and concentrated by the twodimensional centrifugation technique as described above by using microbanding centrifuge tubes, the viruses are amenable for use in many other assays.” (Page 17, line 7). Among the many different types of assays enumerated in the patent, also mass spectrometric analysis with ionization by matrix-assisted laser desorption is mentioned, describing shortly the smearing process onto sample plates with adding matrix solution, according to a cited literature publication. This patent publication is an outstanding description of the various centrifuging procedures which can be applied to body fluids to receive separated species of microbes in complex mixtures of microbes. Interestingly, the claims are directed only to methods and apparatuses to detect and localize light emitted or scattered by the microbanded samples in a centrifuge tube in order to detect the microbands which are difficult to observe.
The objective of the invention is to provide a method with which infectious pathogens in body fluids can be identified preferably within only about one hour.